The solid transport media is a small size card that allows fast, easy DNA extraction from a variety of biological samples. In 2016 we developed a solid media transport card; for that pilot study to control the self-collection we used a pseudo-self-collection technique. The current study expands this prior work using true self-collections and only the POI card, and aims to evaluate the solid media transport card to detect HR-HPV in self-samples compared to liquid transport media.Ten thousand eight hundred eighty-five women between the ages of 30–59 with no screening for 3 years were enrolled. The self-collected sample was first applied to a new solid media transport card (Labeled as SC) then the brush placed in 6 ml ThinPrep liquid (Labeled as SL). Then a physician collected a direct endocervical specimen into ThinPrep liquid (Labeled as DL). Samples were tested with Cobas 4800 and the SeqHPV NGS assay for HR-HPV. Patients positive on any test were recalled for colposcopy and biopsy.p = 1.00).Ten thousand three hundred thirty-nine participants had complete data. The mean age was 43.9 years. CIN 2+ rates were 1.4% (142/10339). The agreement in HPV detection between the two different self-sample collection media was also good . Tested with Cobas, the sensitivity of Cobas-SL and Cobas-SC for CIN 2+ was95.07 and 94.37%; and for CIN3+ was 96.30, 96.30% respectively. The specificity of Cobas-SC, and Cobas-SL for CIN2+ was 88.74 and 87.35%; for CIN3 was 88.04and 86.65% respectively. Tested with SeqHPV, the sensitivity for CIN2+ of Seq-SC and Seq-SL was 95.77 and 96.48%; for CIN3+, both the SC and SL specimens had a sensitivity of 100%. The specificity for CIN2+ of Seq-SC and Seq-SL was 89.54 and 89.53%; for CIN3+ was 88.84,88.82% respectively. For both HR-HPV assays, the sensitivities were similar for the two self-sample media (SC vs SL, The solid transport card for collecting vaginal self-samples as accurate as liquid transport media assayed by two different PCR based HR-HPV tests. The solid transport media is a suitable medium for collecting and storing vaginal self-samples.The online version contains supplementary material available at 10.1186/s13027-020-00333-4. Traditionally cervical swabs have been placed in liquid media for transport. Due to the logistical difficulties such as spillage, flammability, and weight, adding to the risks and costs of liquid transport media, solid carriers consisting of chemically treated or untreated filter paper have been investigated for hrHPV testing. These filter paper cards are easy and safe to store and transport –13. ReceThe Cobas 4800 HPV test, the first approved HPV assay for primary screening by US FDA, is a qualitative multiplex assay, providing specific genotyping information for HPV types 16 and 18, and then 12 other high-risk HPV types as a pooled result . Our stuBetween Aug 2016 to Aug 2018, a multi-center, population-based cross-sectional cervical cancer screening study [Chinese Multi-Center Screening Trial (CHIMUST)] was conducted in China.10,885 subjects were recruited from 15 screening sites in 7 provinces. Women in those sites were eligible if they were 25–59 years of age, sex-experienced but not pregnant, had no cervical cancer screening for at least 3 years, no prior hysterectomy, and no prior pelvic radiation. The study protocol was approved by the human subject review boards of the Peking University Shenzhen Hospital (PUSH), Shenzhen, China, and the Cleveland Clinic, Cleveland, USA. In addition, the study was registered with the Chinese Clinic Trial Registry (chiCTR-EOC-16008456), an international clinical trials registry platform approved by the WHO. Women with incomplete data were excluded from the analyses. This included: (1) women testing positive by any HPV test without having colposcopy and biopsies; (2) Specimen is not sufficient or no sample for an HPV test; (3) Failure of any HPV test.Every woman had contributed two specimens, one was collected by herself (Self-Sample), and one was collected from the endocervix by a clinician (Direct-Sample). The self-collected sample was first applied to the solid media transport card (labeled as SC-sample), then the brush placed in 6 ml ThinPrep® PreservCyt® Solution  (labeled as SL-sample). The brush was then agitated in 6 ml PreservCyt Liquid (as a split sample). Self-sampling instructions were provided by poster diagrams and personal instruction. The physician-collected samples were placed in 20 ml ThinPrep® PreservCyt® Solution (labeled as DL-sample). All samples were stored at room temperature and were tested with Cobas 4800 HPV test and SeqHPV test for HR-HPV within 2 months of collection. Patients testing HPV positive (self or direct), were recalled for colposcopy with directed and random 4 quadrants micro-biopsies plus endocervical curettage (ECC). Histology slides were interpreted by a gynecologic pathologist from PUSH (Author C.W). Immunochemical staining with p16 was selectively obtained to adjudicate difficult cases.The new solid transport card consists of PK 226® paper  treated with a combination of a lysing solution and a dye . The lysing solution contains an ionic detergent to lyse cell membranes and stabilize DNA as well. Similar, to the FTA card, the indicating dye changes color when the sample is applied. The sample area on the card was punched using a 5-mm Harris micro-punch . Each card was manually punched 3 times and placed in a single well in a 96-well plate. Then they were all washed once using 100 μl of sterile water. The water is carefully removed with a sterile fine-tip pipette. The DNA elution is performed with 50 μl of sterile water at 56 °C for 30 min immediately followed by 95 °C for 15 min. in a heating block. The 96-well plate containing DNA elution and pieces of card are then centrifuged at 4000 rpm for 3 min and the eluted DNA is transferred into a new 96-well plate. When performing the assays, 5ul of DNA will be used in each well of the 96 well plates for PCR, for Cobas assay, and SeqHPV. Our prior study shows that 5ul is the standard volume used for the Cobas assay, and SeqHPV, and it has been thoroughly tested and demonstrated to be optimal with > 99% adequate specimens. (Any storage for future use will be at − 80 °C) , 15.All samples were tested with Cobas 4800 HPV test and SeqHPV test for HR-HPV according to the manufacturer’s instructions. All the DL-Samples and SL-samples accepted by the PUSH lab were split, after thoroughly mixed by shaking, 1 cc used for Cobas 4800 and 1 cc for SeqHPV testing. After splitting, the physician-collected samples were processed for cytology interpretation. The Cobas 4800 system platform , consists of the Cobas × 480 instrument and the Cobas z480 analyzer. We used two different specimen preparation procedures for the Cobas z480 that had previously been optimized. Nucleic acids used for the Cobas assay from the two liquid specimens (DL and SL), were prepared using Cobas × 480. The instrument could yield 50 μL of nucleic acid, eluted from 500 μL of ThinPrep solution used per subject. The eluted DNA solutions from the solid cards were prepared according to the Cobas4800 device “special instructions”. The “PCRONLY” program was performed using Cobas z480.5 μL DNA solution from the solid card samples were needed to ensure a sufficient sample (adequate DNA) was present for valid detection.The “split sample” methodology we used, was used extensively in the 1990’s for the development of liquid based cytology . The BotCytology using the Hologic I2 imager systems (computer assisted diagnosis) will be used for future research not for patient care in the current study.Data were entered in an ACCESS database specially designed for CHIMUST. To evaluate the effectiveness of different sample collection media for vaginal self-samples in population-based screening, we calculated sensitivities for detecting CIN2+ and CIN3+, the concordance in detecting high-risk HPV, and the differences in HPV assay performance using solid (filter paper cards) and liquid based specimen transport for the self-collected and physician collected cervico-vaginal specimens. Sensitivity and specificity of the Cobas and SeqHPV testing results in detecting high-risk HPV and CIN2 + were calculated using CIN2+ as the endpoint. McNemar’s Chi-square was performed to calculate differences between paired proportions at a probability level of 0.05. Agreement between self and direct samples was measured by absolute agreement and Kappa statistics (Cohen’s Kappa). Agreement between the solid transport media and liquid transport media for detecting HR-HPV in vaginal self-samples was also measured. All data were analyzed using SPSS 17.0.Ten thousand three hundred ninety-nine women had complete data. Mean age was 43.9 years. The return rate for colposcopy was 81.0%. 1.4% (141 patients) had CIN2+ and 0.5% (or 54 patients) had CIN3 + .101 (0.93%) women were dropped from the analysis due to HPV test failure. 6 (0.05%), 2 (0.02%), 29 (0.27%),34 (0.31%), 19 (0.17), and 19 (0.17) were missing Cobas-DL, Cobas-SL, Cobas-SC, Seq HPV-DL, SeqHPV-SL and SeqHPV-SC, respectively. Testing failure in both the assays were reported by the lab as inadequate DNA , and 13.7% for Cobas and 11.6% for SeqHPV in self-samples placed in liquid medium (labeled as SL-sample), and 12.4% for Cobas and 11.6% for SeqHPV in self-samples collected on the solid transport card (labeled as SC-sample) . The card (SC) was significantly more specific than the self-liquid sample (SL) on the Cobas assay but similar with SeqHPV for both CIN2+ and CIN3+  and the SL (liquid) samples. Our data shows that the POI Card performed well in this self-collection trial demonstrating equal sensitivity to liquid samples run both on Cobas and SeqHPV. We know from our prior work in SPOCCS III, that self-collected specimens will identify more HPV than direct endocervical specimens. This excess HPV found in the vagina is unassociated with CIN in the cervix . Of inteIn 2016 we published our work developing a solid media transport card . The newAfter developing the POI card, we published a second study comparing liquid (SurePath) vs solid media (iFTA and POI cards) with Cobas. For this pilot study to control the self-collection we used a pseudo-self-collection technique  . We wereIn a study from Sweden evaluating the HRVIR assay (laboratory developed test) compared to Cobas using FTA cards, the clinical sensitivity of the FTA card and HPVIR test was equivalent to Cobas, and the Cobas assay detected 63 of 67 women with CIN2+ [94.0% (95% CI = 85.2–98.1)] . LikewisIn our trial when split samples were used, we have twice encountered discordant results, once with the primary card sample and once with the secondary liquid sample. Not with standing, we believe the strength of our work and the current literature supports the equivalency of solid media specimen transport, and liquid transport methodologies. The potential impact for non-liquid specimen transport on mass population-based screening is profound. Especially in current times of COVID, where self-collection methodologies will be preferable than bringing large populations together for move conventional cervical cancer screening events.We believe that the current study adds considerable power to the literature on specimen media comparisons, with 141 cases of CIN2+. The use of non-liquid forms of specimen transport, especially integrated with self-collection sampling screening programs appears to demonstrate equivalency and is an important area for future study and implementation.Additional file 1: Table supplement 1. Rate of HPV testing failure.


In this work, which is part of a larger research program, a framework called “virtual data fusion” was developed to provide an automated and consistent crack detection method that allows for the cross-comparison of results from large quantities of X-ray computed tomography (CT) data. A partial implementation of this method in a custom program was developed for use in research focused on crack quantification in alkali-silica reaction (ASR)-sensitive concrete aggregates. During the CT image processing, a series of image analyses tailored for detecting specific, individual crack-like characteristics were completed. The results of these analyses were then “fused” in order to identify crack-like objects within the images with much higher accuracy than that yielded by any individual image analysis procedure. The results of this strategy demonstrated the success of the program in effectively identifying crack-like structures and quantifying characteristics, such as surface area and volume. The results demonstrated that the source of aggregate has a very significant impact on the amount of internal cracking, even when the mineralogical characteristics remain very similar. River gravels, for instance, were found to contain significantly higher levels of internal cracking than quarried stone aggregates of the same mineralogical type. Despite decades of research, the problem of harmful alkali-silica reaction (ASR) in the field of concrete construction has not yet been satisfactorily solved. For the first time in 1940, Stanton ,3 develo2 within aggregates reacts with alkalis  in the presence of water to form expansive alkali silicate hydrates (ASR gels). Because the tensile strength of road surface concrete is often significantly lower than the swelling pressures caused by ASR gels, cracking can be induced . Al. Al44]. For the categories GK1 and GK4, the selected stones were sieved from a natural river gravel. Such river gravels are typically characterized by significant mineralogical deterioration due to naturally occurring weathering processes. In order to characterize these river gravels, which have a heterogeneous mineralogical composition, the primary types of rock  occurring in GK1 and GK4 were determined and 10 individual grains of each type were selected for CT analysis.For the categories GK2 and GK3, the selected stones were sieved from crushed stone chips that were quarried from solid rock deposits. Such quarried stones are typically characterized by greater mineralogical integrity than river gravels because they have not been exposed to significant weathering. Due to the homogeneity of the quarried stone, the selection of individual grains was limited to 10 each for GK2 and GK3. Thus, combining all the individual grains from each of the four stone categories included in this research study, a total of 90 different individual grains were investigated.During this research program, an acceleration voltage of 130 kV and current of 180 µA were used for the X-ray source. The X-ray beam was also filtered using a 0.5 mm thick Copper plate immediately upon leaving the source in order to remove (unwanted) photons of small energies from the X-ray beam, thereby increasing the contrast of the resulting images. The flat panel detector used for this scanning contained a 2048 × 2048 pixel field.Individual aggregates were sorted based on mineralogical characteristics and placed within corresponding plastic tubes with small pieces of foam separating the aggregates from one another. As a result of heating and deterioration of the target material within the X-ray tube as well as changes in detector sensitivity over time, significant variations in the measured X-ray beam intensity and distribution can occur. To compensate for these variations, “dark-field” and “bright-field” images, which correspond to blank images  acquired with no illumination and full illumination, respectively, were acquired prior to the scanning of each plastic tube of samples. These images were then used to calibrate the X-ray images of the samples.The CT machine was pre-programmed to collect a complete scan of each aggregate before repositioning the plastic tube using a manipulator and beginning the scan of the next aggregate. Thus, the scanning conditions for all stones within any given plastic tube were identical. All scanning conditions other than resolution were also held constant for all plastic tubes.It was important to maintain scanning conditions that were as consistent as possible to ensure that the results of the crack analysis would be comparable. In spite of this, the resolution was maximised for each stone type, if they significantly varied in size and shape. This was done by adjusting the distance between the plastic tube and the X-ray source. The corresponding voxel sizes were then calculated directly from the measured distances between the X-ray source, the sample holder and the X-ray detector for each individual set of scans.Although these variations in image resolution are known to directly affect measurements of crack properties, such as surface area , some estimation of the magnitude of this effect can already be accounted for based on a recent study . The resIn such an environment, where many analysis methods are readily available, but no individual method is sufficiently accurate to provide the needed measurements, the use of a data-fusion inspired approach becomes very promising and attractive. Data fusion enables researchers to combine data from multiple sources in order to produce more consistent and accurate results than those provided by any single source . In non-It is, however, often the case that researchers only possess meaningful data from a single measurement technique, such as CT (often indeed used as a reference), or do not have access to further non-destructive testing equipment. Even in this case, it should be possible to use the theory behind the data fusion approach to improve the overall quality of the quantitative determinations resulting from image analysis. Rather than relying on a wide variety of physical measurement techniques, data fusion in this approach would be carried out by using the output from an array of different image analysis techniques. Since such an approach relies on the fusion of different computationally generated data sources resulting from the same original CT image rather than on a variety of different physical measurement techniques, it can be more appropriately described as virtual data fusion. This is similar to the process that is thought to occur when an expert identifies cracks using the human eye. The expert has only one measurement technique , but by considering its many different aspects , the expert is able to quite easily and accurately detect a crack on the surface of a specimen.Although, in theory, twelve or more different analysis methods could be used in the virtual data fusion implementation for analysing the ASR-sensitive aggregate dataset, in practice such a full implementation was impractical. We selected a partial implementation of the scheme including only three analysis approaches. The choice included the approaches that would produce cracking data with sufficient accuracy for this specific application while simultaneously testing the effectiveness of the virtual data fusion concept for obtaining accurate and consistent results. A successful demonstration of the virtual data fusion method with only three analysis components would then give justification for the further development of the algorithm to gradually include additional analysis modules, simultaneously growing in accuracy and resiliency. Moreover, the modular architecture of the strategy could more easily be adapted to different problems than a full but rigid analysis.In fact, for the specific ASR-sensitive aggregate analysis described in this paper, it was not important to separate internal pores from internal cracks since the surface area of both pores and cracks was vulnerable to ASR degradation. Thus, modules related to this differentiation could be left out of the analysis. Furthermore, since scans were not available at varying levels of degradation, all methods relying on changes in sample state relative to time (such as DVC-based methods) were also left out of this analysis. All image analysis described in this paper was completed using custom algorithms developed and implemented in MATLAB .When cracks or pores have widths exceeding two voxels, the voxels in their centres are completely filled with air. Thus, these central voxels are characterized by a particularly low greyscale value. In order to separate these voxels, a threshold has to be selected and subsequently used for image binarization. All voxels darker than the threshold would then be transformed to white and all voxels lighter than the threshold transformed to black. Given that this threshold must be consistently applied for a large range of datasets, an automated selection method was implemented.Using the triangle selection algorithm ,51, a viWhen an object in a CT-image has either a much higher or a much lower greyscale value compared to the surrounding material, its edges can be identified as regions of high gradient. For this purpose, a gradient-magnitude image is calculated from the original CT-image through the use of the Sobel operator  52]. Si. Si52]. Prior to noise identification and removal, the results from Modules 1 and 2 were combined into a single image . This enIt was found that the use of smaller cubes than 125 voxels (with five voxel long sides) tended to leave considerable noise within the image while the use of larger cubes resulted in the loss of a considerable number of voxels along the path of the cracks. Given that the full CT images each contained over seven billion voxels, such an object with a volume of 125 voxels represented less than one ten-millionth of the total image volume. A flowchart of the entire crack-detection process is provided in After the cracks and internal voids were identified within each of the aggregates, their characteristics could be quantified and compared. Of primary interest for this analysis was the determination of volume and surface area characteristics. In particular, in order to compare the results of the CT-analysis with those of other non-destructive measurement techniques, it was important to separate surface-connected cracks and voids (referred here to as “open voids”) from internally isolated cracks and voids (referred to here as “closed voids”). This is because measurements of internal surface area using the Brunauer–Emmett–Teller (BET) method ,54 only The crack/pore surface area measurements obtained using this approach are provided for all of the river-gravel type aggregates in Error bars in From It is also clear from This research clearly demonstrates the need for universal, automated, and consistent crack detection methods that allow the cross comparison of results from large quantities of CT-scan data from different sample types. A framework, called “virtual data fusion“, was developed that has the potential to successfully provide such a method. A partial implementation of this method in a custom program was developed for use in research focused on crack measurement in ASR-sensitive aggregates. Our results demonstrated the success of the program in effectively identifying crack-like structures and measuring their characteristics such as crack extension (relative surface area) and surface connectivity.These results demonstrate the significant impact that the source of extraction can have on the characteristics of aggregates. Even for aggregates of the same mineral type, river gravels contain significantly higher levels of internal porosity and cracking than quarried stone. This is thought to result from the aggressive weathering process that river gravel is subjected to prior to its selection and use for construction. This indicates that the selection of high-quality aggregate based on mineralogical characteristics alone may be insufficient. It is also clear from these results that there is a significant amount of variability in internal porosity and cracking even for stones with the same mineralogical characteristics and extraction source. Thus, large statistical samples will be necessary to properly characterize each stone type for ASR sensitivity.


To describe characteristics of the vitreomacular interface (VMI) in traumatic macular holes (TMH) compared to idiopathic macular holes (IMH) using immunofluorescence and electron microscopy, and to correlate with clinical data.For immunocytochemical and ultrastructural analyses, premacular tissue with internal limiting membrane (ILM) and epiretinal membrane (ERM) was harvested during vitrectomy from 5 eyes with TMH and 5 eyes with IMH. All specimens were processed as flat mounts for phase-contrast microscopy, interference and fluorescence microscopy, and transmission electron microscopy (TEM). Primary antibodies were used against microglial and macroglial cells. Clinical data was retrospectively evaluated.Surgically excised premacular tissue of eyes with TMH showed a less pronounced positive immunoreactivity for anti-glutamine synthetase, anti-vimentin and anti-IBA1 compared to eyes with IMH. Cell nuclei staining of the flat-mounted specimens as well as TEM presented a lower cell count in eyes with TMH compared to IMH. All detected cells were found on the vitreal side of the ILM. No collagen fibrils were seen in specimens of TMH. According to patients’ age, intraoperative data as well as spectral-domain optical coherence tomography (SD-OCT) analysis revealed an attached posterior vitreous in the majority of TMH cases (60%), whereas all eyes with IMH presented posterior vitreous detachment.The vitreomacular interface in TMH and IMH shows significant differences. In TMH, glial cells are a rare finding on the vitreal side of the ILM. The traumatic macular hole (TMH) is a rare full-thickness retinal tissue defect of the fovea with an interruption of all neural retinal layers leading to severe vision loss and includes 5–8.2% of all various types of macular holes –3. TraumUnlike in idiopathic macular holes (IMH), spontaneous closure is common in TMH. In the literature, the spontaneous closure rates vary between 10 and 50% , 12–20. The pathogenesis of TMH is still under discussion. The most common theory implies that anteroposterior compression and equatorial expansion of the globe appear to result in retinal stress, which may result in a full-thickness macular defect without loss of foveal retinal tissue , 8, 9. AHowever, there is less information about cellular components at the vitreomacular interface in these traumatic types of macular holes. To our knowledge, there is only one case series with one analysed TMH specimen presenting positive immunostaining of glial fibrillary acidic protein (GFAP) and neuron-specific enolase (NSE) . ImmunosPrevious studies showed an attached posterior vitreous body in the majority of eyes with TMH using SD-OCT and clinical examination , 10, 23.The aim of our study was to firstly identify vitreomacular interface changes in surgically excised premacular tissue of eyes with TMH by immunocytochemistry and TEM and then compare the results with the findings in IMH. For clinical correlation, demographic data and SD-OCT findings of both entities were retrospectively analysed.In this clinicopathological study, surgically excised premacular tissue of internal limiting membrane (ILM) and epiretinal membrane (ERM) from eyes with TMH (5 eyes) and eyes with IMH (5 eyes) was consecutively obtained by three different surgeons during vitrectomy at the Ludwig-Maximilians-University, Department of Ophthalmology, between April 2016 and January 2020. All 10 specimens were processed as flat mounts for phase contrast, interference and immunocytochemistry. All 10 specimens were also processed for ultrastructural cell analysis using transmission electron microscopy (TEM). Based on the size of the macular hole diameter using preoperative spectral-domain optical coherence tomography (SD-OCT), control specimens of IMH were selected comparable to specimens of TMH. Clinical data as well as SD-OCT scans using Heidelberg Spectralis OCT  were retrospectively analysed.The Institutional Review Board and the Ethics Committee of the Ludwig-Maximilians-University, Munich, approved the surgical removal as well as the histopathologic preparation and analysis of the patients’ specimens (No. 471–14). Informed consent was obtained from each patient. The study was conducted according to the tenets of the Declaration of Helsinki.The included patients presented with a reduced visual acuity and full-thickness macular holes on biomicroscopy and showed a full-thickness macular defect on SD-OCT. All patients with TMH reported an ocular trauma shortly before the reduction of visual acuity. Besides, typical SD-OCT characteristics for TMH, such as eccentric, ellipsoid-shaped full-thickness macular defects, a large basal macular hole diameter, little to no intraretinal fluid or additional defects like rupture of retinal pigment epithelium (RPE), were found , 10, 23.Patients’ charts were reviewed for age, sex, preoperative and postoperative best corrected visual acuity (BCVA), period of time between diagnosis and surgery, and the follow-up period Table . Using SThe performed surgical technique was a standard 23 gauge pars plana vitrectomy with sequential ILM peeling using end-gripping forceps. For membrane peeling, a vital dye of 0.25 mg/mL solution of Brilliant Blue  was used. At the end of surgery, the vitreous cavity was filled with a tamponade of either air, 20% diluted SF6 or 16% diluted C2F6. For transfer, the tissue was kept in a balanced salt solution (BSS) .After fixation, specimens were flattened and unfolded showing the maximum surface area using a stereomicroscope . Antifading mounting medium 4′,6-diamidino-2-phenylindole  was used to stain the cell nuclei. Primary antibodies were used to identify retinal microglial cells with anti-ionised calcium-binding adaptor molecule 1 (anti-IBA 1)  and macroglial cells with anti-glutamine synthetase (anti-GS)  and anti-vimentin (anti-VIM)  as listed in Table Preparing negative controls, all specimens were dissected into pieces and the primary antibody was substituted with both diluent and isotype controls . All other procedures were identical to the procedures described above.For ultrastructural analysis, dehydration in graded concentrations of ethanol and embedding in Epon 812 were performed directly after post fixation with 2% osmium tetroxide . For staining of the semi-thin sections of 400 nm, an aqueous mixture of 1% toluidine blue and 2% sodium borax was used. Ultrathin series sections of 60 nm followed and were contrasted with uranyl acetate and lead citrate. Using a Zeiss light microscope and a Zeiss EM 9 S-2 electron microscope , 5 grids (each with 8–10 ultrathin sections) per specimen were analysed.In this interventional laboratory investigation, we included surgically excised premacular tissue of ILM and ERM from 4 women (1 eye with TMH and 3 eyes with IMH) and 6 men (4 eyes with TMH and 2 eyes with IMH), corresponding to 5 right and 5 left eyes. The clinical data as well as the analysed SD-OCT characteristics are given in Table The patients’ mean age at the time of surgery was 39.0 ± 29.3 SD years  in TMH and 70.8 ± 7.8 SD years  in IMH. The mean macular hole diameter was 448.8 ± 139.2 SD µm in TMH and 379.6 ± 27.9 SD µm in IMH. The vitreous was attached to the posterior pole in 3 of 5 eyes (60%) with TMH. One eye with TMH showed a complete PVD (20%) and one eye showed an incomplete PVD (20%). All eyes with IMH showed a PVD, of which a complete PVD was seen in 2 eyes (40%) and an incomplete PVD in 3 eyes (60%). The mean period of the time between diagnosis of macular hole and vitreoretinal surgery was 30.3 ± 6.8 SD months  in eyes with TMH and 2.6 ± 2.3 SD months  in eyes with IMH.2F6. TMH showed a primary closure rate of 60% and IMH 80%, respectively. Patient 2 required a second vitrectomy in order to close the TMH. Table The BCVA of patients with TMH was 0.8 ± 0.3 SD LogMAR preoperatively  and 0.7 ± 0.4 SD LogMAR at last follow-up examination . Patients with IMH showed a preoperative BCVA of 0.9 ± 0.1 SD LogMAR  and a BCVA at last follow-up examination of 0.8 ± 0.7 SD LogMAR . One patient was lost to follow-up and had a BCVA of 2.0 LogMAR 2 days after surgery with 90% of the vitreous cavity filled with CThe immunocytochemical analysis as well as the cell count and area of each specimen are shown in Table 2  and in eyes with IMH 1.4 ± 0.7 SD mm2 .In summary, eyes with TMH showed a mean cell count of 39.2 ± 49 SD . Eyes with IMH presented with a mean cell count of 164.4 ± 186.4 SD . The mean surface area in eyes with TMH was 3.4 ± 2.3 SD mmIn the majority of eyes with TMH (3 of 5 eyes), positive immunoreactivity for the microglial cell marker anti-IBA 1 was seen. For macroglial cells, anti-GS was documented positive in 2 of 5 eyes and anti-VIM in 1 of 5 eyes. All eyes with IMH (5 of 5 eyes) revealed a positive immunoreactivity for anti-IBA 1. For anti-GS, 5 of 5 eyes and, for anti-VIM, 4 of 5 eyes with IMH showed a positive immunostaining. Negative controls revealed no immunoreactivity. Figure Colocalisations were seen in both entities and are listed in Table Ultrastructure was analysed in all 10 specimens using TEM. Table The ILM was found in all analysed specimens Fig.  and was In 3 of 5 specimens with TMH, single-layered glial cells were found at the VMI by series sections of the TEM Fig. . An exemIn contrast, specimens of IMH showed multi-layered cells as well as cell clusters on the vitreal side of the ILM as illustrated in Fig. This clinicopathological study presents different immunocytochemical and ultrastructural findings at the VMI analysing surgically excised premacular tissue of ILM and ERM in eyes with TMH and IMH. Compared to a higher cell count and multi-layered cell composition in eyes with IMH, the fibrocellular composition shown in eyes with TMH revealed few single cells without tractive properties.By immunocytochemistry, both entities presented a positive immunoreactivity for the macroglial cell markers anti-VIM and anti-GS as well as for the microglial cell marker anti-IBA 1. However, in eyes with TMH, a large variation of the cellular elements was seen. In general, a positive immunoreactivity of the used glial cell markers was found more often in eyes with IMH. In accordance, transmission electron microscopy (TEM) showed significantly more cellular structures identified as hyalocytes, astrocytes or myofibroblasts in specimens of IMH than of TMH.To our knowledge, there is only one case series investigating the ILM in various maculopathies, among others an eye with TMH . Kanavi Macroglial cells such as Mueller cells and astrocytes are neuronal cells and of a neuroectodermal embryonic origin. In contrast, microglial cells are non-neuronal cells with a mesodermal origin. They originate from macrophages that invade the brain during early development and have macrophage activity . MicroglMacroglia is identified by immunocytochemistry using several markers such as anti-GFAP, anti-VIM and anti-GS –40. AntiIn our study, all three analysed primary antibodies were tested positive in eyes with TMH and IMH. One specimen of TMH revealed no cells, so that immunocytochemistry could not be evaluated (No. 4). Another specimen of TMH only showed 5 cells, which were not detected with positive immunoreaction (No. 1). In IMH, positive immunoreactivity was seen more frequently in all specimens. These findings might allow the conclusion that macroglial and microglial cells are involved in the clinical course after TMH and IMH development. This is supported by the findings of colocalisations. Colocalisation of anti-VIM and anti-IBA 1 was found in 2 of 5 specimens of TMH, which might give evidence of the presence of activated microglia. In specimens with IMH, no colocalisation of anti-VIM and anti-IBA 1 was seen. The colocalisation of anti-VIM and anti-GS that was detected in some specimens of IMH and TMH might indicate the presence of Mueller cells. Unexpectedly, both entities showed the colocalisation of anti-GS and anti-IBA 1. Whether this colocalisation indicates the potential of microglia to endogenously express GS or demonstrates microglia that have previously phagocytised GS-positive cell debris still remains unclear. Though, microglia is not yet known to express the macroglia marker anti-GS.In the literature, there is only one other known TEM case series that describes the analysis of ultrastructural details of the VMI of eyes with TMH, namely by Kumar and colleagues . They idFurthermore, clinical data of the patients in our study revealed that the vitreous was attached to the posterior pole in the majority of eyes (60%) with TMH. However, it remains unclear whether PVD was induced by the ocular trauma or had already existed beforehand as the 2 patients  with preoperative PVD were patients older than 65 years. A high rate of an attached posterior vitreous body is in concordance with findings of previous studies. Huang and colleagues noted that the vitreous was still attached to the fovea in all included patients with TMH . YanagiyAccording to current data, there is no established therapy for TMH. In some cases, surgery might be needed, and in others, the TMH closes spontaneously. In fact, TMH do have a relatively high rate of spontaneous closure up to 50% within the first 9 months after development , 20, 48.On the other hand, it is known that TMH persisting for more than 1 year are less likely to close spontaneously . The recIf surgery is needed, vitrectomy with fluid-gas exchange, gas tamponade and postoperative prone positioning, with or without ILM peeling or ILM flap, with adjunctive agents like TGFβ2 or autologous platelet concentrate and TMH closure using amnion-patches are all methods that showed positive results concerning TMH closure and BCVA gains in several studies , 56–63.Limitations of our study include its relatively small number of analysed specimens, its retrospective analysis of clinical data and the variable period of follow-up. Furthermore, the collection of premacular tissue during pars plana vitrectomy might have been incomplete and could have led to incorrect numbers of cell count measurements. Additionally, it must be noted that due to the preparation of flat-mounted membranes, we were only able to test 3 different primary antibodies and decided to focus on glial cell markers. Comparing the cell count found via immunocytochemistry and TEM, it is likely that not every cell was cut through ultrathin serious sections of 60 nm for TEM. Therefore, we did not focus on statistical analysis and correlation of ultrastructural findings and functional parameters.In conclusion, our results present less cells on the vitreal side of the ILM in eyes with TMH compared to multi-layered cell composition in eyes with IMH. Furthermore, we found a positive immunoreactivity for macro- and microglial cell markers in both entities. Clinical data revealed an attached vitreous body in the majority of eyes with TMH. Vitreous collagen was only detected in specimens of IMH but not in specimens of TMH, suggesting that an incomplete posterior vitreous detachment with vitreoschisis plays no role in the development of TMH.


Hollow nerve guidance conduits are approved for clinical use for defect lengths of up to 3 cm. This is because also in pre-clinical evaluation they are less effective in the support of nerve regeneration over critical defect lengths. Hydrogel luminal fillers are thought to improve the regeneration outcome by providing an optimized matrix inside bioartificial nerve grafts. We evaluated here a modified hyaluronic acid-laminin-hydrogel (M-HAL) as luminal filler for two clinically approved hollow nerve guides. Collagen-based and chitosan-based nerve guides were filled with M-HAL in two different concentrations and the regeneration outcome comprehensively studied in the acute repair rat sciatic nerve 15 mm critical defect size model. Autologous nerve graft (ANG) repair served as gold-standard control. At 120 days post-surgery, all ANG rats demonstrated electrodiagnostically detectable motor recovery. Both concentrations of the hydrogel luminal filler induced improved regeneration outcome over empty nerve guides. However, neither combination with collagen- nor chitosan-based nerve guides resulted in functional recovery comparable to the ANG repair. In contrast to our previous studies, we demonstrate here that M-HAL slightly improved the overall performance of either empty nerve guide type in the critical defect size model.  Peripheral nerve injures (PNIs) arising from trauma with a 2–3% incidence have a strong impact on patients due to their accompanying sensory and motor function loss; for example, dysesthesia, paralysis, and neuropathic pain . Althoug® Nerve Guide for which a large body of pre-clinical and clinical data demonstrate its value as an off-the-shelf product ; PL CMAP AUC = 2.11 ± 0.28 [ms * mV].We further analyzed the recorded CMAPs for quantitative parameters, e.g., their CMAP amplitude area (AUC), from which we also calculated the percentile loss of functional axons in comparison to the contralateral non-lesioned nerve, and their CMAP amplitude ratio, displaying the degree of recovery in relation to the non-lesioned side. The amplitude area of the recorded CMAPs reflects the number of functional axons reinnervating the target tissue, and can therefore be regarded as a sign of nerve recovery. As a reference, the CMAP AUC of the TA , we have not again investigated in the current study, experimental groups we have already been investigating before in the same model . However, for a more comprehensive evaluation of the new data derived from the current study, we want to report specific previously published data again ,31. ThesWith regard to recovery rates, in both previous studies and in accordance to the current study, the ANG repair supported recovery of evocable TA muscle CMAPs in all animals (100%) from 60 days post-surgery onward ,31. RecoAccordingly, data from axon loss calculation demonstrate the same differences as described before for axons innervating the TA muscle. The results are depicted in With regard to recovery of PL muscle function, recovery rates after ANG treatment displayed a similar outcome in both previous studies ,31. Recon = 5; NNG: n = 6; NNG + 0.4% M-HAL: n = 5; NNG + 0.7% M-HAL: n = 6; CNG + 0.4% M-HAL: n = 5; CNG + 0.7% M-HAL: n = 5.The assessment of functional motor recovery by noninvasive electrodiagnostic recordings was supported by the determination of the respective muscle weight ratios (MWRs) of the largest lower hind limb muscles. A high MWR, approximating 1, indicates a recovery in muscle mass due to successful target muscle reinnervation. Therefore, the anterior tibial (TA) muscle and gastrocnemius (GC) muscle were harvested at the 120 days endpoint of the observation period. The ratio between the muscle weights of the treated  limb in comparison to the non-lesioned  limb was calculated. Please note that the muscle weight of two animals was accidentally not recorded and that two animals died during the course of the electrodiagnostic measurements at 60 days post-surgery. Despite this, all animals reaching the endpoint of the observation period, were included in this analysis: ANG: Results from the evaluation of MWRs are graphically depicted in Non-lesioned, contralateral nerve segments, and segments dissected distal from the graft of the reconstructed sciatic nerve were harvested after 120 days, at termination of the observation period. The segments were, fixed, stained, embedded, and sliced into semi-thin cross-sections for histomorphometrical analysis. Stereological analysis was only performed, if the fiber density of the section was high enough to ensure a valid analysis with the described method (see section Nerve Histomorphometry). Consequently the number of analyzed samples did not for all groups match the number of evaluated animals.Since the cross-sectional areas of the samples are not well comparable with one another, the nerve fiber density is considered in the following. As depicted in g-ratio  in two different concentrations. The rationale for adding hydrogel luminal fillers into otherwise hollow nerve guidance channels is based on the fact that empty nerve conduits provide sufficient clinical outcome only when applied for short nerve gap repairs . Both, l® Nerve Guide [® Nerve Guide [® Nerve Guides (NNG) as control and compared all results from the current study with more control conditions, which we have recently evaluated in the same model. Repetition of these control conditions were avoided for ethical reasons. These conditions were likewise ANG repair in both previous studies [® Nerve Guides (emptyCNG) [Therefore, we chose two clinically approved nerve guidance conduits, NeuraGenve Guide  and Reaxve Guide . We haveve Guide ,28,30. R studies , and empmptyCNG) , or CNG Electrodiagnostic recordings of evocable compound action muscle potentials (CMAPs) from muscles at more proximal and more distal locations distal to the lesion site give indications about the temporal progress of functional motor recovery . Here, wFrom analyzing the PL muscle function , it can In comparison to our previous studies, we can summarize that reconstruction of the 15 mm sciatic nerve gap with empty collagen-based  demonstrated a slightly better support than repair with empty chitosan-based  nerve gAs already mentioned above, PL muscle function cannot be considered fully developed at 120 days after surgery for M-HAL associated treatments and comparison to results from our previous studies would rather be a non-appropriate overestimation. It appears noteworthy, however, that adding 0.7% M-HAL was superior in supporting PL muscle recovery over 0.4% M-HAL in the current study. The percentile content of HA in our laminin-hyaluronic acid-based hydrogel luminal filler is influencing the viscosity or stiffness of the luminal content provided to invading cells and regenerating axons. From many studies, it can be concluded that optimally tuned nerve graft mechanical properties can give optimal support to repair Schwann cells as well as pro-regenerative macrophages as well as regenerating axons ,26. Whil2) recovered to a degree comparable to that seen in ANG-treated nerves , but the cell density after three days in vitro (DIV 3) was not significantly different from that in the other conditions anymore. This indicates that as soon as the cells get arranged in M-HAL, they survive and proliferate. Furthermore, this finding demonstrates again that initially cell-free luminal filler hydrogels need to provide mechanical properties and in vivo degradation behavior that would allow timely invasion of regeneration promoting cells into the bioarticial grafts [gndf, fgf-2, and ngf and also gene expression of sox2 and c-jun, transcription factors typically elevated in repair Schwann cells [bdnf showed a trend to be slightly elevated in the 0.4% M-HAL condition than in the other three groups, although the difference was not statistically significant. We can only speculate that expression of brain derived neurotrophic factor, BDNF, has also been elevated within the M-HAL composite bioartificial nerve guides in vivo, and may have accounted for the better outcome in regeneration of myelinated functional motor axons discussed above. Indeed, BDNF plays a role as myelination factor in peripheral nerve regeneration [Luminal filler hydrogels may serve as a cell carrier system for introducing regeneration supporting cells directly together with the bioarticial nerve graft . In thisin vitro , DIV 3 wl grafts . With rel grafts ,20,30. Fnn cells ,40 was rneration .At this point, it seems appropriate commenting on the fact 0.7% M-HAL did not provide optimal properties for our in vitro Schwann cell cultures, but was still evaluated and supportive of axonal regeneration in vivo. We have experienced before that another hydrogel perfectly supporting Schwann cell and even dorsal root ganglion neurite outgrowth in vitro was not at all supportive to axonal regeneration in vivo . TherefoWith regard to a recent report describing more pronounced beneficial effects of hyaluronic acid-laminin hydrogel in a delayed repair approach for 25 mm tibial nerve defects in the rabbit , we wantFrom the current study, we can, at first, conclude that the achievable benefit of adding 0.4% or 0.7% M-HAL as luminal filler, to otherwise empty nerve guides, did not significantly differ with regard to the material the bioartificial nerve graft was made of. Therefore, both collagen- or chitosan-based nerve guides appear to be equally suitable for a combination with M-HAL luminal filler hydrogel. Second, both concentrations of M-HAL provided a better support for axonal and functional recovery than 0.2% HAL in chitosan-based nerve guides before . Finallyw/v) or 0.7% (w/v) hyaluronic acid  [w/v) and 0.7% (w/v) HA solutions were stored at 4 °C. The gel was stored on ice until use. M-HAL was prepared prior to use, based on 10 μg/mL laminin , and 0.4%  . HA stocThe animal experiments were conducted in accordance with the German animal protection regulations and European Communities Directive 2010/63/EU for the protection of animals for experimental purposes. The experiments were previously approved by the Animal Care Committee of Lower Saxony, Germany  in accordance with the local Institutional Animal Care and Research Advisory.p < 0.05, and type II error: β: 0.2, power 50%.In this study, we used 36 young adult female Lewis rats , which were obtained from Janvier Labs SAS  at an age of 10 weeks. Group size was calculated by power analysis with Graphpad Statmate 2.0 software , based on data from previous investigation of another hydrogel , with ef®, Neuraxpharm Arzneimittel GmbH, Langenfeld, Germany) from two weeks ahead of the surgery until the study was completed, to prevent self-mutilation [During the experiment, the animals were housed in groups of four in an enriched environment, under uniform housing conditions . Food and water were provided ad libitum. The drinking water was supplemented with amitriptyline hydrochloride .All surgeries were performed on 13-week old rats under deep anesthesia  and aseptic conditions. Throughout the surgery, the animals were placed on a heating pad to mitigate the decrease in body temperature. Additionally, a heating lamp was used during anesthesia induction and recovery phase. Rectal body temperature was measured before and after surgery in order to ensure a body temperature not lower than 36.5 °C. The recovery phase was supported by applying 2 mL of electrolyte solution  subcutaneously. A sufficient level of analgesia was achieved by subcutaneous injection at a dosage of 0.5 mg/kg Butorphanol  and lidocaine  were topically applied on the exposed sciatic nerve 5 min before nerve transection, in order to ensure sufficient analgesia. Using a micro scissor, the first incision was applied 5 mm distal to the gluteus muscle’s aponeurosis. A 15 mm nerve defect was established for autologous nerve grafts (ANGs). The nerve graft was flipped over, rotated 180°, and sutured distal and proximal, each with three epineural 9-0 stitches spaced approximately 120° apart. A 13 mm nerve defect was established in all experimental groups and reconstructed with a 19 mm artificial nerve graft, generating a 2 mm overlap of both nerve ends  and Reaxon® Nerve Guides  were preconditioned in 0.9% sodium chloride rinsing solution  for at least 20 min. Before tying the knot at the distal suture of the artificial nerve grafts, 60–120 µL of M-HAL luminal filler were applied. Wounds were closed with 3–4 resorbable single button sutures in the femoral biceps muscle  followed by 3–4 nonresorbable mattress sutures  for skin closure.Ahead of luminal filling, NeuraGenFor ethical and animal welfare reasons , we have not investigated again in the current study experimental groups we have already investigated before in the same model . However, we used the previously obtained data for a more comprehensive evaluation of our new data derived from the current study. These previously obtained data have been published in Stößel et al. 2018 , and in The progress of functional motor recovery was monitored by serial non-invasive electrophysiological measurements 60, 90, and 120 days after nerve reconstruction. The muscle weight ratio (MWR) for lower hind limb muscles was determined after completion of the 120-day observation period. All surgery was performed by KHT and evaluators of regeneration progress were blinded to the conditions of sciatic nerve reconstruction applied.Assessment of functional motor recovery with transcutaneous electrodiagnostic recordings: ® Keypoint® Focus device; Natus Europe GmbH, Planegg, Germany) was used. The electrodiagnostic managements were performed in accordance with previously published studies of our group [For the evaluation of ongoing muscle reinnervation 60, 90, and 120 days post-surgery, a portable electrodiagnostic device  = AUChealthy − AUCregenerated/AUChealthy × 100. For the statistical analyses of electrodiagnostic measurements , all animals reaching the endpoint of the analysis were considered. Two animals died due to anesthesia complications after the first functional evaluation at 60 days post-surgery (ANG-group and CNG + 0.4% M-HAL-group).Functional motor recovery was described qualitatively in terms of recovery rates (%) that were calculated from the number of animals per group displaying evocable CMAPs. Quantitative parameters were derived by analyzing the area under the curve (AUC) of the negative peaks of the CMAP Values for CMAP amplitude area and amplitude ratio were set to 0 for certain animals, which did not show evocable CMAPs, whereas the respective values for axon loss were set to 100%. Muscle weight ratio: After the last electrophysiological recording at 120 days post-surgery and harvest of nerve tissue for histology (see below), the anaesthetized animals were sacrificed in carbon dioxide atmosphere and by cervical dislocation after deep anesthesia was induced. The gastrocnemius (GC) and TA muscle of both hind limbs were excised and weighed. The MWR was calculated dividing the muscle weight of the lesioned side by the muscle weight of the non-lesioned side.n = 3). The dissected nerve segments were transferred in Karnovsky fixative  for 24 h. Afterwards, the samples were rinsed with 0.1 M sodium cacodylate buffer supplemented with 7.5% sucrose and post-fixed in 1% osmium tetroxide for 1.5 h. Staining of the myelin sheath was performed in 1% potassium dichromate (24 h), 25% ethanol (24 h), and hematoxylin  [Histomorphometrical analysis of the regenerated nerves was performed in accordance with a previously described protocol . The samor 24 h) . SubsequThe stereological evaluation was performed according to our previously published work ,31,45. In = 3; ANG: n = 5; NNG: n = 4; NNG + 0.4% M-HAL: n = 6; NNG + 0.7% M-HAL: n = 6; CNG + 0.4% M-HAL: n = 4; CNG + 0.7% M-HAL: n = 5. One sample from the CNG + 0.7% M-HAL was further excluded from consecutive nerve morphometrical analysis, because it was not showing an appropriate number of regenerated myelinated axons. Stereological analysis was only performed if the fiber density of the section was high enough to ensure a valid analysis with the described method. Consequently the number of analyzed samples did not for all groups match the number of evaluated animals: non-lesioned control: g-ratio plug-in [g-ratio. Depending on the regeneration of the sample, a total number of at least thirty to a maximum of eighty axons per animal was evaluated. The data was collected under the assumption that the axons show a circular shape. Samples that showed no quantifiable axonal regeneration in the stereological analysis were excluded from morphometrical analysis. For each animal, six to eight view fields (in 100× magnification) of regions showing a high fiber density were acquired to analyze the morphometry of the regenerated nerves. ImageJ  extended by a  plug-in ,31,47 wa2 in a humidified atmosphere. PLL coating was performed by covering the culture dishes with PLL for 45 min at room temperature. After removing the PLL, plates were washed 2 times with Ampuwa® . To prevent excessive fibroblast contamination, 1 mM of arabinoside-c  was added at 2 and 3 days in vitro, respectively. The cells were finally purified to >90% by immunopanning, purity was controlled in immunocytochemistry, and cells propagated in medium as described above, just with an increased concentration of 2 µM Forskolin [Schwann cell cultures at passage 11 were taken from storage in liquid nitrogen and thawed as required. These cells were previously derived from primary neonatal Schwann cells (SCs) obtained from Wistar RjHan:WI rat pups and cultured and purified following a previously published protocol  and in aorskolin . Neonatal rat SCs were cultured in a six-well plate  containing the above described medium (see section Primary Schwann cell culture), serving as control. The initial cell density was 300,000 cells per well. Experimental conditions were cultured within 0.2% HA, 0.4% HA, or 0.4% M-HAL. We excluded 0.7% M-HAL culture conditions from this analysis, because Schwann cells could not be suspended gently enough in this concentration. Further, appropriate amounts of total RNA could not be extracted. Medium was used for preparing the needed concentrations. The respective solutions were prepared one day ahead, as described above (see section 0.4% M-HAL and 0.7% M-HAL preparation), except that the HA was dissolved in culture medium instead of PBS, vortexed thoroughly and stored overnight at 4 °C. The 0.4% M-HAL was prepared freshly. TM cDNA Synthesis Kit . The qRT-PCR was performed according to a previously describes protocol [T-values were calculated with StepOne-software version 2.3 using a constant cycle threshold of 0.2. Calculation of relative amount of transcript in cDNA levels was performed and normalized to the housekeeping gene peptidylprolyl isomerase A (ppia). The primer sequences are given in Total RNA of 3 wells was analyzed after 3 days in vitro as described previously . In brieprotocol . The reaStatistical analysis of data obtained from the current study was performed using Excel Version 2010  and GraphPad Prism version 8.01 . Normal distribution of all datasets was examined with Shapiro-Wilk test. If normal distribution was given, one-way study of variation (ANOVA) with Tukey’s multiple comparisons post-hoc test was used to identify significant differences. If normal distribution was rejected, the data was subjected to Kruskal–Wallis test with Dunn’s multiple comparisons post-hoc test was applied. A two-way ANOVA with Tukey’s multiple comparisons post-hoc test was used to compare the electrodiagnostic recordings during the observation period. All results are presented as percentages or mean ± SEM, as indicated in the respective tables or figures.p < 0.05; two symbols = p < 0.01; three symbols = p < 0.001. Statistical significance was defined and indicated as follows: one symbol =


Aspergillus spp. are spore forming molds; a subset of which are clinically relevant to humans and can cause significant morbidity and mortality. A. fumigatus causes chronic infection in patients with chronic lung disease such as asthma, chronic obstructive pulmonary disease (COPD) and cystic fibrosis (CF). In patients with CF, A. fumigatus infection can lead to allergic disease, such as allergic bronchopulmonary aspergillosis (ABPA) which is associated with high rates of hospitalizations for acute exacerbations and lower lung function. ABPA results from TH2 immune response to Aspergillus antigens produced during hyphal growth, marked by high levels of IgE and eosinophil activation. Clinically, patients with ABPA experience difficulty breathing; exacerbations of disease and are at high risk for bronchiectasis and lung fibrosis. Oral corticosteroids are used to manage aspects of the inflammatory response and antifungal agents are used to reduce fungal burden and lower the exposure to fungal antigens. As the appreciation for the severity of fungal infections has grown, new therapies have emerged that aim to improve treatment and outcomes for patients with CF. Aspergillus spp. are ubiquitous spore forming molds, a subset of which are clinically relevant to humans and can cause significant morbidity and mortality. Pulmonary infection from A. fumigatus, the most common Aspergillus pathogen, causes a diverse set of diseases, ranging from acute invasive disease to long-term, chronic infections [fections . The typfections . The morfections ,4 and prfections . PatientAspergillus can result from stable active infection of the lung or from allergic sensitization resulting from the exposure to Aspergillus antigens. In the first case, disease results from stable and persistent infection of the airways with Aspergillus resulting in fungal growth and an inflammatory response that aims to clear the infection from the lung. In some instances, this has been referred to aspergillus bronchitis [H2-driven immune response to Aspergillus antigens, include both severe asthma with fungal sensitization (SAFS) and allergic bronchopulmonary aspergillosis (ABPA). Both SAFS and ABPA are significant clinical issues in patients with asthma, with the latter being a significant clinical problem in patients with CF [A. fumigatus is the common cause of ABPA, however sensitization to other Aspergillus species has been noted [Aspergillus species. It has been suggested that there may be a continuum of disease that starts with aspergillus bronchitis and progresses to sensitization and ultimately ABPA [Chronic diseases caused by onchitis , which m with CF ,8. A. fuen noted . In the ely ABPA , for theAspergillus colonization and persistence may vary among patients and require continued characterization, allergic fungal infections have a clear deleterious clinical impact. CF patients with sensitization to A. fumigatus antigens have a distinct and robust TH2 inflammatory response in sputum samples after allergen challenge. This inflammation is marked by increases in sputum eosinophils and increased expression of IL-5 and IL-13 [H2 hypersensitivity reaction in response to fungal antigens that drives immune cell activation and eosinophil recruitment  samples with similar prevalence and at similar ages as the common the bacterial colonizers Staphylococcus aureus, Pseudomonas aeruginosa, and Haemophilus influenzae. However, whereas S. aureus and P. aeruginosa prevalence decreased over time due to antibiotic therapy, Aspergillus prevalence remained unchanged [. flavus ,19,20. Totic use ,22. Moreotic use ,24,25. Inchanged . Aspergillus colonization and persistence has increased in recent years. Several studies have suggested an association between A. fumigatus infection, respiratory function and severe pulmonary exacerbations. In several studies, CF patients with chronic A. fumigatus infection have lower percent predicted forced expiratory volume (FEV1) than uninfected controls [Aspergillus were associated with air-trapping on HRCT, although the same study failed to show an association with lung function decline between the ages of 5 and 14 [Aspergillus culture positivity to changes in structural lung disease [Aspergillus infection was associated with worse initial CT scores that declined further in the subsequent year, with the most significant impact on air-trapping and mucus plugging [Aspergillus infections over the course of the study, suggesting a dose-responsive relationship between infection and disease. A similar association with progression of lung diseases was observed for P. aeruginosa infection, but not for S. aureus and H. influenzae infections. An appreciation for the clinical significance of controls , higher controls , show mocontrols ,26,27 ancontrols . Declinecontrols ,30, whic5 and 14 . A recenAspergillus are required criteria together with at least two additional supportive features: eosinophilia (>500 cells/μL), Aspergillus-specific IgG, and/or radiographic findings [An understanding of incidence and prevalence is further hampered by the difficulty of diagnosing disease. Diagnostic criteria for ABPA include both obligatory and supportive criteria in addition to having either asthma or CF. High levels of serum IgE (>1000 U/mL) and a positive hypersensitivity skin test or increased IgE antibody to findings . ABPA isfindings .Aspergillus detection compared to molecular methods, such as quantitative PCR [Aspergillus in sputum using RT-PCR and high-volume culture techniques have the potential to significantly increase the sensitivity of detection and ultimately, diagnosis [Aspergillus, those with ABPA and those with aspergillus bronchitis [The range of reported disease prevalence and variations in diagnostic approaches suggests that pulmonary fungal infections in CF may be under diagnosed. This is further complicated by the use of culture-based diagnostic methods, which underestimate tive PCR ,33. New iagnosis ,34. Usinonchitis . Using tonchitis . They foonchitis .P. aeruginosa and Stenotrophomonas maltophilia infections are positively correlated with Aspergillus infections [Burkholderia cepacia complex was negatively associated with both current and future Aspergillus infection [Aspergillus infections. Potential interactions between Aspergillus species and non-tuberculous mycobacterial (NTM) infections have not been well characterized. Given the importance of NTM infections in CF [A. fumigatus can negatively impact NTM infection in mice [CF patients have a complex lung microbiota, where there is likely significant interplay between colonizing bacteria and fungi . Longitufections . In contnfection , indicatns in CF  and the  in mice , a more P. aeruginosa and A. fumigatus, respectively [Aspergillus colonization [In CF patients, the most common bacterial and fungal isolates are ectively  and coloectively ,41. Givenization . P. aeruginosa and A. fumigatus form biofilms in vivo and in vitro [P. aeruginosa inhibits A. fumigatus planktonic growth and biofilm formation by secreted factors and different isolates of P. aeruginosa exhibit different degrees of toxicity [P. aeruginosa inhibits A. fumigatus growth by the production of pyoverdine, a siderophore that sequesters iron. P. aeruginosa mutants defective in pyoverdine production are not toxic to A. fumigatus, and the addition of pyoverdine to mutant cultures restores A. fumigatus toxicity [Both in vitro ,43. Sevetoxicity ,44. P. atoxicity . More reS. maltophilia is a Gram-negative pathogen of increasing significance in CF. Data from an in vitro mixed-culture biofilm model of A. fumigatus and S. maltophilia suggest an inhibitory effect of S. maltophilia on A. fumigatus growth and production of extracellular matrix [A. fumigatus to amphotericin B was increased in mixed-culture biofilms, whereas S. maltophilia susceptibility to levofloxacin decreased [A. fumigatus and bacteria other than P. aeruginosa. Further study of interactions between A. fumigatus and bacteria commonly found in the CF patients is warranted. r matrix . Co-cultecreased . These dAspergillus from the airways to reduce antigen stimulation of the allergic response [In addition to managing the symptoms of asthma or CF, treatments targeted at treating ABPA aim to prevent acute exacerbations, reduce pulmonary inflammation and to prevent progression toward end-stage fibrotic disease . While tresponse . Theraperesponse ,49. Imprresponse ,51. CurrThe use of corticosteroids in treating ABPA in asthma has largely been based on experience in clinical practice with few randomized, controlled clinical trials studying steroid use as chronic therapy. Long-term steroid use is associated with adverse side-effects, which must be managed in parallel with the management of ABPA ,57,58 anAspergillus infection in the airway and the resulting allergic inflammatory response that is the hallmark of ABPA inflammation. A high percentage of asthmatics sensitized to A. fumigatus are sputum culture-positive for A. fumigatus growing in their airways [Aspergillus-specific IgG and IgE [Aspergillus infection in patients with ABPA and SAFS, 9 patients that were positive for Aspergillus infection by PCR became negative for Aspergillus infection following treatment with itraconazole. This conversion was associated with a reduction in total serum IgE [Use of antifungals in management of ABPA is supported by a strong biological link between  airways , which c airways . Fungal  airways ,62,63, h and IgE . Likewiserum IgE . Aspergillus bronchitis [cyp51A gene, the molecular target of azole activity, the development of resistance to one azole can result in broad cross-resistance to multiple azoles [cyp51B, a second 14-α sterol demethylase, which may be further exacerbated by a second mutation in hmg1 [The most common antifungal therapy used in the management of ABPA is itraconazole, a triazole that inhibits fungal cytochrome P450 synthesis of ergosterol, a critical component of the fungal cell wall . Clinicaonchitis  and ABPAonchitis ,54,55,68onchitis . As withonchitis . Of parte azoles . This co in hmg1 . Oral itraconazole efficacy in asthmatics with ABPA has been studied in two randomized, placebo-controlled studies to study the clinical response and anti-inflammatory effect of treatment ,54. In aA. fumigatus antigen exposure is the main driver of clinical disease. In a second randomized, double-blind placebo-controlled study the effect of itraconazole on pulmonary inflammation was assessed in 29 subjects with stable ABPA [Aspergillus antigens, were also reduced [p = 0.007), itraconazole had a significant benefit to the majority of patients, with fewer side effects than steroid treatment [Inflammation resulting from ble ABPA . Over 16 reduced . More rereatment . Although anti-fungal drugs have not been widely studied in CF patients with ABPA, data generated in asthmatics suggests that antifungal therapy may provide benefit to CF ABPA patients. This is further supported by small studies of itraconazole in patients with CF. In a study of itraconazole in six ABPA patients, three of whom had CF, itraconazole treatment reduced steroid use and two of the three CF patients had clinical benefit, including improved lung function . An addiAmphotericin B, a polyene anti-fungal that acts by disruption of the fungal cell wall, is commonly used as an intravenous drug to treat severe fungal infections in immunocompromised patients . In an eElevated serum and sputum IgE levels are a hallmark of ABPA. IgE can trigger mast cell degranulation and cause hypersensitivity responses in the lung, which together drive the pathophysiology of the disease . OmalizuA. fumigatus [Despite the advances in diagnosis and management of ABPA, there remains a significant unmet medical need for the treatment of ABPA. The primary antifungal therapy, oral itraconazole, is generally safe and well tolerated in both CF and non-CF patients, though there is an extensive list of drug-drug interactions (DDI), which requires drug monitoring during therapy. Itraconazole absorption and pharmacokinetics can be highly variable, resulting in inconsistent exposure across patients, which may impact the consistency of clinical responses ,72. In humigatus . Variablumigatus . A. fumigatus. In addition, PC945, a novel azole delivered by liquid nebulization is also in development as a therapy for treating pulmonary fungal infections  is a dry powder formulation of itraconazole being developed using a proprietary inhaled delivery technology called iSPERSE . A PhaseZP-059  is a dry powder formulation of voriconazole being developed using a novel spray drying technology for the treatment of ABPA in asthma . This foTFF-VORI  is a dry powder formulation of voriconazole formulated using thin film freezing technology, which produces excipient-free nanoaggregates of drug for inhalation ,95. A PhA. fumigatus [PC945  is a novel triazole being developed for liquid nebulization for the treatment of IPA, with potential for use in ABPA. PC945 is a potent inhibitor of ergosterol synthesis, exhibiting 14-fold greater potency than voriconazole and 2.6-fold more potency than posaconazole against umigatus . A Phaseumigatus . Aspergillus-related allergic diseases in patients with CF. With this understanding, more work is needed to decipher the relationship between A. fumigatus infections and different clinical outcomes. An increased appreciation of the clinical significance of ABPA has led to an understanding of the importance of the interactions between fungal and bacterial infections. Additional research in these areas is warranted to further characterize the complex microbial ecology of the CF lung and to help identify new treatment strategies for the management of disease. In recent years there have been several large, well-controlled clinical studies of therapies for ABPA, which have significantly improved treatments for patients and established a framework for the continued study of new therapies in development. The assessment of anti-fungal drugs with novel mechanisms of action as treatments for ABPA and other allergic fungal diseases would be a welcome step towards improving patient lives. Significant advances have been made in understanding the incidence and severity of